KR20040004374A - Hair nourishments and method of screening the same - Google Patents

Abstract

A wool agent comprising at least two kinds of inhibitors for 5α reductase type 2, inhibitors for transforming growth factor β2, and inhibitors for caspase-3; And screening for at least two types of inhibition of inhibition to 5α reductase type 2, inhibition to transforming growth factor β2 and inhibition to caspase-3, or screened for inhibition to caspase-3, Next, there is provided a method characterized by screening for the inhibition of apoptosis, and a hair loss inhibitor having an inhibitory action against caspase-3 and a hair loss having an inhibitory action against caspase-3 and an apoptosis inhibitory action. Provide inhibitors.

Description

HOL NOURISHMENTS AND METHOD OF SCREENING THE SAME}

Male hair loss is known to involve male hormones and apoptosis. In general, when a signal of apoptosis is transmitted to a cell, a cascade composed of a plurality of caspases is activated, and at the downstream end, caspase-3 is generated in precursor caspase-3, which is a cytoskeletal protein. It is also known that by cleaving ICAD (Inhibitor of Caspase-Activated DNase), the cell leads to irreversible death (apoptosis).

However, the hair loss process ranging from the involvement of male hormones to apoptosis by caspase has not been elucidated, and the relationship between hair loss and caspase-3 has not been elucidated. Therefore, a reasonably designed wool agent based on the clarification of the hair loss apparatus is not known.

The hair cosmetics, called the wool or hair growth agents, are expected to promote hair extension. The wool or hair cosmetics penetrate the hair roots to expand blood vessels and promote blood circulation. It contains a drug that stimulates the papilla of the hair, assists in the production of hair, and promotes hair growth. When the agent contained in the wool is expressed by action, blood circulation promotion, local irritation, follicle revival, hair male hormone, anti seborrhea, keratin dissolution, sterilization or anti-inflammatory The component which shows an effect is mentioned.

Specific examples of ingredients conventionally formulated in anticipation of the above action include senburi extract, vitamin E, red pepper periodicity, vitamin B ₆ , hinokitiol, estradiol, sulfur, salicylic acid, menthol (Menthol), glycyrrhizin, or pantothenic acid.

As the method for evaluating the wool, a method using an animal such as a mouse, a rabbit or a monkey, a method using a human, a method using a tissue culture, and the like exist as existing methods. Among them, the method of using a human is a direct method of observing the hair extension by applying a test substance to the human head, etc. The method of using a tissue culture, for example, the presence of the test substance in the hair follicles separated from the skin. There is a method of measuring the elongation of hair cultured by culturing under.

In these conventional methods, the method using the former human requires a long time to obtain the result, and the latter method requires a microscope to obtain the result, and the operation is complicated, and a plurality of samples at once There are problems such as difficulty in testing. For this reason, there has been a demand for a screening method that can be performed in a short time and with simple operation, and that a large number of samples can be assayed at one time.

Therefore, the present inventors have applied for the screening of wool, further developed a novel screening method that can test a plurality of samples at a time and with a simple operation and has already applied for a patent (Japanese Patent Application No. Hei 11-86406) .

TECHNICAL FIELD The present invention relates to a wool agent and a screening method thereof, and more particularly, to a wool agent containing a hair growth phase extension material of a hair cycle in a combination of two or more kinds thereof, and a method for selecting the same. will be.

In addition, the present invention relates to a caspase-3 inhibitor, and more particularly, to a caspase inhibitor which has a hair growth period extension activity of the hair cycle and contains a plant extract as an active ingredient, and the same. It relates to wool agents, in particular hair loss inhibitors.

The present invention also relates to a composition for the prophylaxis or treatment of hair or the like containing isocoumarins, particularly phyllodulcin, as an active ingredient. More specifically, the present invention relates to a wool feed, hair degenerative transition inhibitor, cirrhosis treatment agent or nephritis treatment agent containing isocoumarins, in particular, pilodulcin as an active ingredient. More suitably, it relates to a wool or hair degenerative transition inhibitor containing isocoumarins, particularly dihydroisocoumarins, in particular filodulcin as active ingredients, which promote hair extension.

1 is an electrophoresis diagram showing that 5αR-II is expressed only in the frontal region of male hair loss patients.

Fig. 2 is a photograph showing that the human hair follicles are promoted to the degenerative phase when organ cultured for 6 days in the presence of TGF-β2.

3 is a photograph showing the distribution of TCF-β2 in the growth phase and the degeneration phase of human hair follicles. Strong staining of TGF-β2 in the arrow part is recognized.

4 is a photograph showing the change (immune staining) of TGF-β2 from the growth phase to the degenerative phase. Immunostaining of TGF-β2 that is strong at the border (arrow) of the dermal papilla and follicles is recognized.

Fig. 5 is a photograph showing the distribution of TGF-β2 in the early stage of the degeneration of human hair follicles.

Fig. 6 is a graph showing the effect of anti-TGF-β2 neutralizing antibody on the hair growth in the case of organ culture of human hair follicles.

Fig. 7 is a graph showing the effect of fetuin on the elongation of hair when organ cultured human hair follicles.

Fig. 8 is a photograph showing that hair cells around the dermal papilla in the degenerative phase are causing apoptosis (activation of caspase-3).

9 is a photograph showing the distribution of precursor caspase-3 over the human parental cycle.

10 is a graph showing the effect of z-VAD-fmk on hair growth in the human hair follicle organ culture method.

FIG. 11 is a photograph showing a change in the shape of a hair bulb in the transition from the growth phase to the degenerative phase.

12 is a graph showing the caspase-3 inhibitory activity of various plant extracts.

13 is a photograph showing that the morphology of the follicles is well maintained in the coexistence of fetuin, a TGF-β2 inhibitor, and Z-VAD-fmk, a caspase-3 inhibitor.

14 shows that apoptosis is suppressed by the addition of quacharrate extract in a system in which Pam212 cells cause apoptosis in the presence of TNF alpha and CHX, and is a photograph showing the morphology of the organism.

Fig. 15 is a photograph showing that activation of precursor caspase-3 is inhibited by addition of quacharrate extract or VAD-fmk in Pam212 cells cultured in the presence of TNFα and CHX.

16 shows the chemical structural formulas of known components in the sag extract.

Fig. 17 shows the results of investigating pilodulcin for the action of inhibiting the expression of PAI-1 by TGF-β2.

18 shows the results of measuring Alamar Blue-reduction rate for cytotoxicity of pilodulcin.

19 shows the results of experiments on the effect of promoting hair follicle elongation on pilodulcin.

It is a photograph which shows the hair irradiation result of hair when filodulcin is added to human isolated hair.

It is a technical object of the present invention to provide a reasonably designed wool agent and a screening method thereof due to the clarification of the hair loss apparatus.

In order to solve the above technical problem, the inventors of the present invention have found that 5α reductase type 2 [5αR-II] is expressed in the frontal region of male hair loss patients, and this enzyme is expressed from testosterone in the frontal region. Produces a more potent male hormone, DHT, which acts on papillary cells to promote the production of transforming growth factor β2 (TGF β2), and TGF β2 activates caspases in follicular epithelial cells to A series of cascades (degenerator cascades) were elucidated to induce sheaths, thereby facilitating the transition from the growth phase to the regression phase in the mother cycle, that is, miniaturization and softening, leading to hair loss.

And the present inventors discovered the novel wool agent and its screening method based on this clarification.

Therefore, the present invention

(1) an inhibitor against 5α reductase type 2 (5αR-II),

(2) an inhibitor against transforming growth factor β2 (TGF β2), and

(3) inhibitors against caspase-3

At least two types of them are mix | blended and the wool agent provided is characterized by the above-mentioned.

(2) an inhibitor against transforming growth factor β2 (TGF β2) located downstream of the cascade; And (3) an inhibitor against caspase-3.

In addition, the present invention

(1) selecting an inhibitor against 5α reductase type 2 (5αR-II),

(2) selecting an inhibitor against transforming growth factor β2 (TGF β2), and

(3) selecting inhibitors for caspase-3,

A method for screening inhibitors for male hair loss characterized by combining at least two kinds of selections is provided.

It is preferable to combine (1) selection of inhibitory substance against transforming growth factor β2 (TGF β2) located downstream of the cascade and (2) selection of inhibitory substance against caspase-3.

However, this method is preferred for the screening of hair loss inhibitors, which is desirable for testing a large number of test substances, but lacks precision for the final selection of the target substance. Therefore, it is preferable to first screen and select a test substance by the above method, and then select a more direct and appropriate apoptosis inhibitor.

Accordingly, the present invention also selects an inhibitory substance against caspase-3, and subsequently selects a substance that inhibits apoptosis in cultured cells of mouse epidermal keratinocytes for the selected substance. Provided are methods of screening inhibitors for hair loss.

It is a technical object of the present invention to provide a novel caspase inhibitor which is useful as a wool agent, in particular a hair loss inhibitor component.

In order to solve the above technical problem, the inventors of the present invention have found that 5α reductase type 2 (5αR-II) promotes the production of testosterone (DHT), and this testosterone is transforming growth factor β2 (TGF β2). ), And TGF β2 activates caspases, thereby exploring a series of cascades that promote the transition from the growth phase to the degenerative stage of the mother cycle, namely, miniaturization and softening, leading to hair loss.

Therefore, the screening of hair loss inhibitors can be performed by any of the selection of inhibitors against caspases, particularly caspase-3, which is present at the bottom of the cascade.

The present invention therefore provides novel inhibitors of caspase-3 and wools, in particular hair loss inhibitors, containing them as active ingredients. As such caspase-3 inhibitors, extracts of indigophera tinctoria Linn., Extracts of southern hemisphere (Nanbansaikachi), jewellery, extracts, tormentira extracts, grape leaves extracts, barley berry extracts , Maple leaf extract, extract of kwacharate (ク ア チ ャ ラ ラ テ), black tea extract, oolong tea extract, yulmu extract and root extract may be exemplified.

However, in the above method, since the hair loss inhibitor merely retains caspase-3 inhibitory activity, it is preferable to have apoptosis inhibitory effect more directly.

Accordingly, the present invention also provides a wool component that retains inhibitory activity against caspase-3 and retains the activity of inhibiting apoptosis in cultured cells of mouse epidermal keratinocytes. As such a component, the quacharrate extract, maple leaf extract, black tea extract, oolong tea extract, yul radish extract, and root root extract can be illustrated.

Another novel screening method of the present invention is that the tumor growth factor (Transforming Growth Factor-β2; TGF-β2) promotes the transition from the growth phase to the degenerative phase in the hair growth cycle, thereby antagonists of TGF-β2. It was developed based on what the present inventors found out that the hair growth effect can be obtained by suppressing the transition to the retrograde.

In addition, the inventors of the present invention, when cultured human papilla cells in the presence or absence of an antagonist to TGF-β2 and TGF-β2, plasminogen-activator inhibitor (PAI-1) produced in the absence of antagonists It was confirmed that the amount of PAI-1 decreased in the presence of TGF-β2 antagonist compared to the amount of). The present invention was developed based on the discovery of this identified novelty.

In the process of confirming the effectiveness of this screening method, screening is carried out on various herbal drugs to confirm that many herbal drugs have an antagonistic action against TGF-β2. Similarly, the patent application. In addition, it was also confirmed that gamchi extract has the strongest antagonistic action against TGF-β2.

Further studies were carried out on the persimmon extracts which proved to have the strongest antagonistic action (TGF-β inhibitory activity with PAI-1 as an indicator, kidney action by human capillary organ culture). . That is, it could be confirmed that isocoumarins, particularly filodulcin, which is one of dihydroisocoumarins, were the main body of the action.

Embodiment of the invention

The growth cycle of human hair is hair loss after 5-6 years of growth, 2-3 weeks of regression and 2-3 months of rest, followed by the development of new hair and its growth phase. First, the inventors have experimentally demonstrated that TGF-β2 induces and initiates the degenerative phase, that is, the growth phase is shortened. In other words, when the growth follicle obtained from the human scalp is organ cultured in the presence or absence of TGF-β2, and cultured in the presence of TGF-β2, morphological changes such as the transition to the degenerative state in a natural state occur. Confirmed.

In addition, the distribution of TGF-β2 in human follicles in the growth phase and human follicles in the anagen phase was observed by immunohistostaining using anti-TGF-β2 antibodies, and TCF- was significantly remarkable in the follicles of the anagen phase compared to the human follicles in the growth phase. It was found that β2 was expressed and distributed (Example 1).

In addition, human hair follicles were organ-cultured in the presence and absence of anti-TGF-β antibody and Fetuin, which are known to be antagonists of TGF-β2, to measure hair height. As a result, it was confirmed that in the presence of anti-TGF-β2 antibody or fetuin, hair growth was larger than in the absence thereof, and TGF-β2 was neutralized by the antagonist of TGF-β2, and the transition to the regression phase was suppressed. (Example 2).

In addition, the present inventors confirmed in Example 3 that apoptotic cells were generated in hair follicle cells when the follicles were degenerated by staining human degenerative follicles by the TUNEL method.

Subsequently, caspase-3 was observed in each phase of the hair cycle, and it was confirmed that caspase-3 existed throughout the hair cycle, suggesting that caspase-3 functions in apoptosis. .

Thus, in organ culture of human blankets, carbobenzoxy-valyl-alanyl-β-methyl-aspart-1-yl-fluoromethane (z-VAD-fmk), which is known to be a caspase-3 inhibitor, is replaced with As hair was added, hair extension and hairball shape retention were observed. As a result, it was confirmed that z-VAD-fmk, a caspase-3 inhibitor, promotes hair growth and is involved in maintaining the morphology of the hair follicle, and the caspase-3 inhibitor inhibits the growth phase in the hair cycle. It was found that the wool effect was exerted.

In view of the above, alopecia is characterized by hyperactivity of testosterone (DHT) by 5αR-II, hyperactivity of TGF β2 by testosterone, activation of caspase by TGF β2 and activation of caspase. It is evident through the cascade of progress.

Therefore, it is preferable that the wool agent for inhibiting hair loss contains two or more types of combinations of an inhibitor of activity on 5αR-II, an inhibitor of action of TGF β2 and caspases, particularly an inhibitor of caspase-3 at the bottom of the cascade. .

In addition, the hair loss inhibiting wool agent is any one of the selection of an activity inhibitor against 5αR-II, a selection of an inhibitor of action of TGF β2 and a selection of an inhibitor against caspases, particularly caspase-3 at the bottom of the cascade. Screening can be carried out by a combination of two or more kinds of selections.

Inhibition of activity of 5αR-II can be measured, for example, in the following manner. 25 μl of enzyme 5αR-II solution, 25 μl of sample and radiotestosterone solution ( ³ H-testosterone (1 × 10 ⁶ dpm), testosterone 1 μM, glucose-6-phosphate (5 mM), glucose-6-phosphate dehydrogenase [ 2 units / ml] and NADP (1 mM) were incubated at 37 ° C. for 40 minutes to stop the reaction, and then the reaction product was separated by thin layer chromatography, and dpm was counted.

Inhibition of the action of TGF β2 can be measured, for example, in the following manner. Using TGF-β2 to induce the production of PA1-1, cells (e.g., dermal papilla cells) were cultured in the coexistence of TGF-β2 with the test substance, and PA1-1 in the medium was measured. It is measured whether the phase change of -1 is suppressed.

Inhibition of caspase-3 can be measured, for example, in the following manner. As a buffer solution for the reaction solution, for example, 25 mM HEPES (pH 7.5) was used, and 10% sucrose and 0.1% of 3-[(3-koramidepropyl) dimethylammonia] -1-propanesulfonate ( CHAPS) and 5 mM dithiositol (DTI). In addition, the fluorescent substrate acetyl-Asp-Glu-Val-Asp-methylcoumarinamide (Ac-DEVD-MCA) synthesized as a substrate is added to a final concentration of 0.5 mM. Subsequently, a test sample was added to the solution, incubated at a constant temperature for a predetermined time (for example, at 37 ° C. for 30 minutes), and the reaction was stopped with a reaction stop solution, followed by a fluorometer (excitation: 355 nm; emission: 460 nm) It can measure by.

The apoptosis inhibition of the present invention can be measured, for example, in the following manner. First, cultured cells of mouse epidermal keratinocytes are prepared. That is, the epidermal keratinocytes of newborn BALB / c mice are cultured in high nutrient medium (DMEM medium) containing 10% fetal bovine serum (FCS) and 5 times the concentration of amino acids and vitamins. As a result, large cells having an unclear cell boundary proliferate predominantly, and a part of the culture surface is multiply layered to produce a high cell density covered with keratin. Large cells with no clear cell boundaries are removed by trypsin treatment, and this operation is repeated over several months to collect a stratified cell population. This is Pam212 cells. At passage, cells are detached and dispersed by 0.05% trypsin and 0.1% EDTA, and the confluent cells are divided into 1:10 and spawned on a new dish. It is fused again.

The morphology has characteristics of keratinocytes cultured in the normal epidermis from normal epidermis. That is, cells having a circular or polygonal shape proliferate while forming colonies in a monolayer. Some tend to be stratified. When it is in an overfusion state, the whole cell layer peels and falls off from a dish in many cases.

It expresses keratin of the same molecular weight (67, 59, 55, 50 kDa) as the neonatal mouse epidermis. Ornithine decarboxylase activity is higher than that of primary cultured epidermal keratinocytes and is markedly enhanced by phorbol ester treatment. Inoculation of newborn mice of the same series has a 100% chance of forming squamous cell carcinoma to obtain proliferative capacity in agar. Epidermal keratinocytes of primary culture are prepared by proliferation and differentiation according to the height of Ca ²⁺ concentration, but the change is not clear in Pam 212. However, in low concentrations of Ca ²⁺ (<0.1 mM), no intercellular bonds are formed, and changes are also seen in the cytoskeleton.

The cells cause apoptosis in the presence of tumor necrosis factor α (TNF α) and cyclohexamide (CHX), resulting in floating dead cells. Therefore, for the screening of apoptosis inhibitors, culture of mouse epidermal keratinocyte cells, such as Pam212 cells, in 10% FCS + DMEM medium containing TNF α (10 ng / ml) and CHX (10 μg / ml) In this case, the cultured state to which the test substance is added and the culture to which the test substance is not added may be conducted to compare the state of generation of floating dead cells.

Human hair growth cycles occur after 5-6 years of growth, 2-3 weeks of regression, and 2-3 months of rest, followed by the development of new hair, which begins its growth. The present inventors first demonstrated experimentally that TGF-β2 induces and initiates the degenerative phase, that is, the growth phase is shortened. In other words, when organ growth of the hair follicles obtained from the human scalp is carried out in the presence and absence of TGF-β2, and cultured in the presence of TGF-β2, morphological changes in the same manner as in the transition to the degenerative state in the natural state occur Confirmed.

In addition, the distribution of TGF-β2 in human follicles in the growth phase and human follicles in the anagen phase was observed by immunohistostaining using anti-TGF-β2 antibody, and TGF- remarkably remarkably in the follicles of the anagen phase compared to human follicles in the growth phase. It was found that β2 was expressed and distributed (Reference Example 1).

In addition, human hair follicles were organ-cultured in the presence and absence of anti-TGF-β antibody and Fetuin, which are known to be antagonists of TGF-β2, to measure hair height. As a result, it was confirmed that in the presence of anti-TGF-β2 antibody or fetuin, hair growth was larger than in the absence thereof, and TGF-β2 was neutralized by the antagonist of TGF-β2, and the transition to the regression phase was suppressed. (Reference Example 2).

In addition, the present inventors confirmed in reference example 3 that apoptosis occurs in hairy cells when hair follicles are retracted by staining human degenerative follicles by the TUNEL method.

Subsequently, when caspase-3 was observed in each phase of the hair cycle, caspase-3 was confirmed to exist throughout the hair cycle, suggesting that caspase-3 functions in apoptosis. It became.

Thus, in organ culture of human blanket, carbobenzoxyl-valyl-alanyl-β-methyl-aspart-1-yl-fluoromethane (z-VAD-fmk), which is known to be a general inhibitory substance, is replaced with As hair was added, hair extension and hairball shape retention were observed. As a result, it was confirmed that z-VAD-fmk, a caspase-3 inhibitory substance, promotes hair extension and is involved in maintaining the shape of the hair follicle portion, and the caspase-3 inhibitory substance extends the growth phase in the mother cycle. It discovered that it exhibits the wool effect.

In view of the above, androgenetic alopecia is apoptosis due to hyperstimulation of male hormone (DHT) by 5αR-II, hyperstimulation of TGF β2 by male hormone, activation of caspase by TGF β2 and activation of caspase. It is evident through the cascade of progression.

Thus, the wool agents of the present invention can be selected as inhibitors for caspases, especially caspase-3, which is the most downstream of the cascade. Examples of such caspase-3 inhibitors include quacharrate extract, maple leaf extract, black tea extract, oolong tea extract, juvenile radish extract, root extract, indigopera tinctoria linn extract, extract of Southern Bansaikachi, and clown Beard extract, tormentira extract, grape leaf extract, and bodhi tree extract.

Inhibition of caspase-3 can be measured, for example, in the following manner. As a buffer for the reaction solution, for example, 25 mM HEPES (pH 7.5) was used, and 10% sucrose and 0.1% of 3-[(3-coramidepropyl) dimethylammonio] -1-propanesulfonate) (CHAPS) and 5 mM dithiothreitol (DTT). In addition, the fluorescent substrate acetyl-Asp-Glu-Val-Asp-methylcoumarinamide (Ac-DEVD-MCA) synthesized as a substrate is added to a final concentration of 0.5 mM. Next, a test sample was added to this solution, incubated at a constant temperature and constant temperature (for example, at 37 ° C. for 30 minutes), and the reaction was stopped with a reaction stop solution, followed by a fluorometer (excitation: 355 nm; emission: 460). nm).

The apoptosis inhibition of the present invention can be measured, for example, in the following manner. First, cultured cells of mouse epidermal keratinocytes are prepared. That is, the epidermal keratinocytes of newborn BALB / c mice are cultured in high nutrient medium (DMEM medium) containing 10% fetal bovine serum (FCS) and 5 times the concentration of amino acids and vitamins.

As a result, large cells with no clear cell boundaries multiply predominantly, but a part of the culture surface has a high density of cells that are stratified and covered with keratin. Large cells with no clear cell boundaries are removed by trypsin treatment, and this operation is repeated over several months to collect a stratified cell population. This is Pam212 cells. At passage, cells are detached and dispersed by 0.05% trypsin and 0.1% EDTA, and the fusion cells are divided into 1:10 and spread on a new dish to reach a fusion state again in 2 to 3 days.

The morphology has characteristics of keratinocytes cultured in the primary culture from normal epidermis. That is, cells having a circular or polygonal shape proliferate while forming colonies on a single layer. Some tend to be stratified. When it is in an overfusion state, the whole cell layer peels and falls off from a dish in many cases.

It expresses keratin of the same molecular weight (67, 59, 55, 50 kDa) as the neonatal mouse epidermis. Ornithine decarboxylase activity is higher than that of primary cultured epidermal keratinocytes and is markedly enhanced by phorbolester treatment. Inoculation of newborn mice of the same system has a 100% chance of forming squamous cell carcinoma to obtain proliferative capacity in agar. Epidermal keratinocytes of primary culture are prepared by proliferation and differentiation by the height of Ca ²⁺ concentration, but the change is not clear in Pam212. However, in low concentrations of Ca ²⁺ (<0.1 mM), no intercellular bonds are formed and a change is also seen in the cytoskeleton.

Such cells cause apoptosis in the presence of tumor necrosis factor α (TNF α) and cyclohexamide (CHX), resulting in floating dead cells. Therefore, for screening of apoptosis inhibitors, cultured mouse epidermal keratinocyte cells, such as Pam212 cells, were cultured in DMEM + 10% FCS medium containing TNF α (10 ng / ml) and CHX (10 μg / ml). In this case, the cultured state to which the test substance is added and the culture to which the test substance is not added may be conducted to compare the state of generation of floating dead cells.

Examples of a hair loss inhibitor having a caspase-3 inhibitory activity and an apoptosis inhibitory activity include quasarate extract, maple leaf extract, black tea extract, oolong tea extract, yul radish extract, root extract, and the like. Larate extracts are particularly preferred.

In order to obtain a plant extract, the extractive plant material is preferably dried and made into small pieces or shredded as necessary. As the extractant, water, an organic liquid, a mixed solvent of an organic solvent miscible with water, and the like can be used, and a mixed solution of an organic solvent miscible with water and water is particularly preferred. Ethanol, methanol, etc. are mentioned as an organic solvent. Preference is given to aqueous ethanol solutions, in particular 70% aqueous ethanol solutions. As extraction concentration, the temperature from room temperature to the boiling point of an extractant can be used, 20-37 degreeC is especially preferable. Extraction takes 3 hours to 7 days.

The extract may be dried by removing the solvent according to a conventional method such as evaporation under reduced pressure, or, if a non-toxic solvent such as water or an aqueous ethanol solution is used as the extraction solvent, the extract may be concentrated as it is or after appropriate concentration. It can be used as a component.

The present invention also relates to a wool powder obtained by the second screening method, a hair degenerative transition inhibitor, a liver cirrhosis treatment agent or a nephritis therapeutic agent, wherein the wool powder contains isocoumarins (compounds), in particular piluludocin as an active ingredient. It is. In addition, all other hair degenerative transition inhibitors, liver cirrhosis drugs, and nephritis drugs also contain isocoumarins (compounds), particularly pilodulcin, as active ingredients.

EMBODIMENT OF THE INVENTION Hereinafter, embodiment of this invention is described. First, the novel screening method which produced this invention is demonstrated concretely.

The growth cycle of human hair is hair loss after 5 to 6 years of growth, 2 to 3 weeks of regression and 2 to 3 months of rest, then new hair develops and the growth phase begins anew.

The present inventors have experimentally demonstrated that TGF-β2 induces and initiates the degeneration phase, that is, the growth phase is shortened. In other words, when organ growth of the hair follicles obtained from the human scalp is carried out in the presence and absence of TGF-β2, and cultured in the presence of TGF-β2, morphological changes such as transition to the degenerative state in a natural state may occur. Confirmed that it occurred. In addition, the distribution of TGF-β2 in human follicles in the growth phase and human follicles in the anagen phase was observed by immunohistostaining using anti-TGF-β2 antibody, and TGF- remarkably remarkably in the follicles of the anagen phase compared to human follicles in the growth phase. It was found that β2 was distributed in expression.

In addition, in the presence and absence of anti-TGF-β-antibody and Fetuin, which are known to be antagonists of TGF-β2, human hair follicles were organ cultured to measure the height of hair. As a result, in the presence of anti-TGF-β2-antibody or fetuin, the hair growth is larger than in the absence of these, and TGF-β2 is neutralized by the antagonist of TGF-β2, whereby transition to the regression phase is suppressed. Was confirmed

Subsequently, in order to find a means of simply causing the antagonist effect on TGF-β2, by culturing the dermal papilla cells in a medium containing TGF-β2 at various concentrations and measuring PAI-1 in the culture, TGF-β2 It was confirmed that the acidity of PAI-1 in the dermal papilla cells was promoted. In addition, in culturing the dermal papilla cells in the presence of TGF-β2, it was found that the increase in the production of PAI-1 is suppressed by adding an anti-TGF-β2-antibody which is an antagonist of TGF-β2. These experiments confirmed that the presence, amount, activity, etc. of the antagonist against TGF-β2 can be easily measured by measuring PAI-1 in the culture of dermal papilla cells according to a conventional method.

Based on the above recognition and discovery, the inventors also developed a screening method. In other words, by culturing the dermal papilla cells in the presence of TGF-β2 and various test substances, the amount of PAI-1 in the culture was measured, and the kidneys were cultured by organ culture of the follicles on the test substance which brought the low PAI-1 measurement. As a result of the measurement, it was confirmed that the test substance for lowering the PAI-1 measurement value promoted the elongation of the hair, and confirmed the effectiveness of this screening method.

According to this method, cells (hair papilla cells, fibroblasts) having PAI-1 generating ability may be cultured in the presence of TGF-β2 and the test substance, and the amount of PAI-1 in the culture may be measured. In that case, if the measured value of PAI-1 is significantly lower than the control without adding the test substance, the test substance is determined to be TGF-β2 (antagonist) and can be a candidate for wool.

The dermal papilla cells used in this method are isolated from human scalp according to the method of Messenger (Messenger AG: BR J Dermatol 110, 685, 1984). In addition, the culture is using Dulbecco's improved Eagle's medium (DMEM) and the like to which 10% of calf serum (FBS) is added. In addition, immortalized cultured papillary cells may be used as the papillary cells to be used. Immortalized dermal papilla cells have the advantage that they can be obtained in large quantities from time to time by their culture, so that a large number of test substances can be screened at one time or several times.

Immortalized dermal papilla cells infect SV40-antigens with normal dermal papilla cells, and after several weeks of culture, many clones of cells from growth-type colonies have been shown to cultivate clones with excellent proliferative capacity and close to normal dermal papilla cells. By continuing to establish immortalized cell lines. For example, it can be established according to the method proposed by Hada et al. (Japanese Patent Application No. Hei 9 -271927).

The dermal papilla cells may be cultured in a commercial medium for culturing animal cells. The concentration of TGF-β2 in the screening medium is approximately 0.01 ng / ml-100 ng / ml, and the culture is usually performed in the presence of CO ₂ at 37 ° C. Incubation time until PAI-1 is measured is approximately 8 to 72 hours.

Fibroblasts can also be isolated and cultured by conventional methods, and immortalized cells can be established. The measurement of PAI-1 is measured according to a conventional method, for example, by sandwich ELISA (enzyme immunoassay) using an anti-PAI-1 antibody.

The present invention confirmed the effectiveness of the woolen hair or hair degenerative transition inhibitor of the present invention by using the screening method developed by the present inventors and confirmed the effectiveness thereof, and the effectiveness of the screening method according to Examples 10-13. Explain.

The present invention provides a sheep's wool, a hair degenerative transition inhibitor, a liver cirrhosis treatment drug or a nephritis therapeutic agent, containing isocoumarins, especially hydroisocoumarins, in particular, hydrodocumarin, especially pilodulcin as an active ingredient, as described above. It can be provided in the form of medicines, quasi-drugs or cosmetics.

The formulation is not particularly limited as long as it can exhibit the effects of the present invention and can be selected arbitrarily. For example, liquid, oil, ointment, cream, gel, aerosol, mousse and the like can be exemplified. Specifically, tonic, lotion, conditioner, scalp treatment, shampoo, rinse and the like can be mentioned.

And in these formulations, the component which can be mix | blended with a pharmaceutical and cosmetics normally can be mix | blended other than the isocoumarins compound containing the pilodulcin which concerns on this invention in the range which does not impair the effect of this invention. For example, examples of the medicinal ingredients include senburi extract, vitamin E and its derivatives, and nicotinic acid esters such as nicotinic acid benzyl ester.

In addition, examples of drugs that promote blood circulation by local stimulatory action include capsicum spermatozoon, Cantharides tincture, camphor, and nonyl acid and nirylamide. Examples of the agent having the follicle activating action include hinokithiol, placental extract, photosensitizer, pantothenic acid and derivatives thereof. Examples of the drug having an anti-male hormone action include hormonal agents such as estradiol, ethynyl estradiol, and estrone. Examples of the drug having an anti-seborrheic effect include sulfur, thiocylon and vitamin B ₆ .

In addition, salicylic acid, resorcin, and the like having keratinizing action and bactericidal action may be used to prevent the occurrence of dandruff, and glycyric acid, derivatives, menthol, and the like may be applied to the blanket to prevent inflammation of the scalp. Amino acids, such as serine, methionine, and arginine, vitamins, such as biotin, herbal extracts, etc. are mentioned for nutrient dissemination and reactivation of enzyme activity.

In addition, Yangwook, Yulmu rice, peppermint, asparagus, red pepper, aloe, goji berry, mugwort, rice, manza, rice flavor, bone clam, gold leaf, gentian, dansam, loofah, bellflower, Pine, Red Ginseng, Angelica, Safflower, Barberry, Arcea Semen, Eucalyptus, Red Pepper, Akebiae Caulis, Wax, Siho, Tea Tree, Cone, Hop, Chrysanthemum, Three You can also combine plant extracts such as Polygala senega L., sesame seeds, thorns, cashews, brown roots, safflower, saffron, rosemary, yogurt, and mallow.

Vasodilation agents such as alkoxycarbonylpyridine N-oxide, calpronium chloride and acetylcholine derivatives; Skin antifungal agents such as cephaanthin; Antibacterial agents such as hexachlorophene, benzalkonium chloride, cetylpyridium chloride, undecylenic acid, trichlorocarbanide, bithionol, phenol and isopropylmethylphenol; Zinc and its derivatives; Lactic acid or alkyl esters thereof; Citric acid; Succinic acid; Organic acids, such as malic acid, and protease inhibitors, such as a tranesamic acid, can also be mix | blended. In addition, drugs such as cyclosporic acid, Oxendolone, diazoquisid, minoxidil, and the like can also be blended.

Moreover, alcohol, such as ethanol and isopropyl alcohol; Polyhydric alcohols such as glycerin, propylene glycol and polyethylene glycol; Oil components such as higher fatty acids, higher alcohols, hydrocarbon oils, natural fats and oils, ester oils and silicone oils; Surfactants; Flavorings: chelating agents; Moisturizing agents such as 1,3-butylene glycol, hyaluronic acid and derivatives thereof, multitor, atherocollagen, sodium lactate; Thickeners such as Quinds oblonga, carboxyvinyl polymer, xanthene gum and the like; High molecular compounds; Antioxidants; Ultraviolet absorbers; Pigments; water; The component normally mix | blended with a pharmaceutical and cosmetics, such as a stabilizer, can be mix | blended.

Isocoumarins, which are active ingredients of the prophylactic or therapeutic composition of the hair of the present invention, can be blended into external preparations for medicines, quasi-drugs, cosmetics, etc., and are particularly useful as therapeutic agents for skin disorders that improve skin disorders such as acne. Do. The form of the external preparation for skin is not particularly limited as long as it is a dosage form usually applied as an external preparation for skin, such as an ointment, lotion, emulsion, cream, pack, gel, foundation, lip balm, face color, skin cleanser, bath solvent, and the like. Moreover, in addition to cowenes, the component which can be mix | blended with a skin external preparation can be mix | blended with the external preparation for skin normally in the range which does not produce a problem.

Moreover, the composition containing the isocoumarins of this invention can be used also as a pharmaceutical composition and cosmetic composition of that excepting the above. The pharmaceutical composition may be any of oral and parenteral formulations, and the formulation may be any, for example, liquid, syrup type, tablet, powder, granule, capsule, suppository or injection.

At the time of the formulation, if necessary, by using conventional additives such as diluents, excipients, binders, disintegrants, lubricants, colorants, colloids, stabilizers, solubilizers, pH preparations, etc. It is good to manufacture. For example, to prepare a liquid formulation, physiological saline, ethanol, 1,3-butylene glycol, or the like can be used as a diluent or carrier.

Although the compounding density | concentration of the isocoumarin compound in the composition of this invention is suitably determined according to the form of a composition, the purpose of use, etc., it is normally mix | blended in 0.001-2 weight% in a composition. If it is less than 0.001% by weight, it is difficult to fully exhibit the effects of the present invention, while if it is more than 2% by weight, it is not preferable in formulation.

In addition, the dosage is appropriately determined depending on the form of the composition, the purpose of use, and the like, but in the case of a woolen or external preparation, usually 0.001 to 100 mg is divided or applied once or several times a day to the scalp or skin. It is desirable to.

The present invention is explained in detail by the following examples.

Example 1

Confirmation that 5αR-II is expressed only in dermal papilla cells obtained from the frontal head of male hair loss patients

The mRNA of cells obtained from the frontal and posterior heads of male hair loss patients was PCR amplified by primers for 5αR-I, 5αR-II, AR and G3PPH amplification, and the results of the detection are shown in FIG. 1. As a result, it was confirmed that 5αR-II is expressed only in the frontal head.

Example 2

Confirmed that TGF-β2 promotes the onset of the retrograde stage

(1) Promotion of retrograde stage by TGF-β2

Three antibiotics of penicillin, streptomycin and Fungizone were added to Williams E medium (Gibco), and 10 μg / ml hydrocortisone, 10 ng / ml insulin, 10 ng / ml sodium selenate and 10 μg, respectively. The medium to which / ml transferrin was added was used for human capillary organ culture using the medium containing insulin and the medium not adding as a basal medium.

Using micro shear blades under stereoscopic microscope, hair follicles of growth phase were isolated from human scalp. Isolated follicles were washed with basal medium and then measured for length, allowed to sink in insulin-containing medium (using 24 well microplates: 1 ml per well) to give a gas phase of air containing 37 ° C. and 5% CO ₂ . Incubated overnight under conditions. After measuring the length again, the blankets with elongation of 0.25 mm or more and the shape of the growth phase were maintained were selected and divided into groups of 10 to 12 so as to make the kidneys even. For each group, after exchange with a medium containing the test substance, the culture was continued under the above gaseous conditions.

The elongation of the blanket was measured over time by inserting a micrometer into the eyepiece portion of the inverted microscope. Photographs of the entirety of the blanket and the ridges were taken by a camera connected to an inverted microscope.

On the second day of culture, TGF-β2 was replaced with a medium in which a final concentration of 50 µg / ml was added to the basal medium, and the culture was continued while observing elongation and morphological changes of the follicles for 5 days. As shown in FIG. 2, the morphology of the degenerative phase was promoted in the follicles to which TGF-β2 was added at 6 days of culture compared to the control group without addition. Other proliferative factors and cytokines were not recognized as promoting categorical morphological changes. Therefore, it was thought that TGF-β2 has a catenary action.

In addition, the change in the form of the blanket when the transition from the growth phase to the regression phase in natural hair is shown in FIG. 10. From the comparison of FIG. 2 and FIG. 10, it is clear that TGF-β2 facilitated the transition to the regressor.

(2) Localization of TGF-β2 in human blanket

Human scalp tissues or organ cultured hair follicles were washed with PBS, fixed with 4% paraformaldehyde-phosphate buffer (pH 7.2) for 4 hours, dehydrated with ethanol series, wrapped with paraffin, and then stained with 5 μm thick tissue. Made a section.

In order to elucidate the role of TGF-β2 in human hair follicles, localization of TGF-β2 in human hair follicles was investigated. Local observation of TGF-β2 in human hair follicles was performed by anti-TGF-β2 antibody immunostaining of human scalp tissue sections.

Using a paraparaffin-treated and ethanol hydrophilic human scalp tissue section, anti-TGF-β2 antibody (Santa Cruz) as the primary antibody, peroxidase-labeled rabbit IgG as the secondary antibody, avidin-biotin- By the peroxidase complex method, the site of presence of TGF-β2 in human hair follicles was immunohistochemically stained. As shown in FIG. 3, TGF-β2 (immunostaining staining) was recognized in the outermost layer of the outer root sheath in the hair follicle at the late growth stage. On the other hand, TGF-β2 (immunostaining staining) was recognized in the hair follicles of the late degenerative phase by the cells of the outermost layer of the outer root sheath and the degenerating epithelial cells of the upper head of the breast milk. From this, it was thought that TGF-β2 is working in late stage of the retrograde stage.

(3) Localization of TGF-β2 in the early stage of the retrograde stage of human blanket

When hair follicles enter the degenerative phase, the proliferation of hair follicles decreases, and then stops, and it is considered that a substance that inhibits the proliferation of hair follicle cells in the early phase of the degeneration phase is acting. TGF-β is known to strongly inhibit the proliferation of epithelial cells, and TGF-β may act in the early stages of degeneration to stop the proliferation of hairy cells and induce degeneration. To verify this possibility, localization of TGF-β in the early stages of the retrograde phase should be made clear.

Although it is very difficult to find the early degenerative follicles in human scalp tissue sections, TGF-β was stained to show the early degenerative changes by analyzing more than 1000 consecutive sections obtained from five alopecia patients. As a result, as shown in FIG. 4, almost no TGF-β2 (immunostaining staining) was recognized in the hair and the nipple of the hair follicles, which had maintained their full growth phase. On the other hand, in the follicles showing a similar morphological change to the degenerative follicles, strong immunostaining of TGF-β2 was recognized at the boundary between the hair and the papilla. From this, it became clear that TGF-β2 acts to induce the degenerative phase.

In the organ culture method of follicles, when the follicle fragments of the growth phase are incubated with an insulin-containing medium, the growth phase morphology is maintained for about 2 weeks, whereas when the follicles are cultured in a basal medium containing no insulin, It is changed to form similar to a blanket. Therefore, by culturing the growth phase follicles in a basal medium containing no insulin, and investigating the localization of TGF-β2 in the follicles showing slight morphological changes, it is possible to estimate whether TGF-β2 is active in the early stage of the retrograde phase. have. Therefore, after organ culture for only one day, the blanket which maintained the form of the growth phase and the blanket which showed the form change similar to that of the degenerative phase were immunostained with the anti-TGF- (beta) 2 antibody. As a result, it was recognized that the change as shown in FIG. 4 actually occurred. This is shown in FIG.

Example 3

Inhibitory Effects of TGF-β2 on Hair Degeneration

As mentioned above, since TGF-β2 acts in the human hair cycle and it is thought that the degeneration phase is induced, it is thought that if the action of TGF-β2 is suppressed in the hair follicles of the growth phase, the transition to the regression phase may be prevented or slowed down. That is, the hair growth phase extension effect by TGF-β2 inhibition can be expected. Specifically, in the case of adding a substance that inhibits the action of TGF-β2 in the human hair follicle organ culture method, the effect of inhibiting the hair degeneration phase was verified by whether the hair extension was promoted or the degenerative shape change was suppressed. .

(1) Effect of TGF-β neutralizing antibody

The inhibitory effect on hair degenerative transition of TGF-β neutralizing antibodies (known to have neutralizing action of human TGF-β1 and TGF-β2) was verified. The method followed the human blanket organ culture method. On day 2 of the culture, a medium in which anti-TGF-β antibody (Genzyme Co., Ltd.) or a mouse IgG of the control group, which had a neutralizing effect of TGF-β, was added to the basal medium to a final concentration of 20 μg / ml or 100 μg / ml. Culture was continued while observing elongation and morphology change of the blankets for 4-7 days. As shown in Figure 6, the addition of TGF-β neutralizing antibody showed a tendency to promote the extension of the hair. In addition, as shown in Table 1, the proportion of the hair follicles in which the morphology of the hair follicles were maintained was increased by the addition of the TGF-β neutralizing antibody.

Proportion of retained blanket Experiment 1 (day 8) Experiment 2 (day 5) Control (mouse IgG) 36.4% (4/11) 55.6% (5/9) 20 μg / ml anti-TGF-β antibody 54.5% (6/11) 100 μg / ml anti-TGF-β antibody 77.8% (7/9)

(2) Influence of cyanosaccharide protein fetuin

The cyrosaccharide protein fetuin (molecular weight 48,400) is a substance present in mammalian fetal serum or in adult blood of various diseases (especially during trauma). In addition, fetuin has a similar amino acid sequence and secondary structure as the receptor of TGF-β and acts as an antagonist of TGF-β ( Demetriou M et al: J Biol Chem 271: 1 2755-12761, 1996 ). The effect of suppressing the transitional stage of phosphorus hair was verified.

Pettuin (final concentration 1, 10, 50 μM) or control bovine serum albumin (final concentration 50 μM) was exchanged with the medium added to the basal medium and the culture continued for 7 days while observing elongation and morphological changes of the follicles. did. As shown in FIG. 7, compared to the bovine serum albumin addition of the control group, fetuin addition significantly promoted hair growth. From the results of these TGF-β neutralizing antibodies and fetuin, the effect of inhibiting hair regression during the TGF-β (2) action was demonstrated.

Example 4

Confirmation of apoptosis in retrograde blanket

Human scalp tissues or organ cultured hair follicles were washed with PBS, fixed with 4% paraformaldehyde-phosphate buffer (pH 7.2) for 4 hours, dehydrated with ethanol series, wrapped with paraffin, and then stained with 5 μm thick tissue. Made an intercept.

When the human degenerative hair follicles are stained by the TUNEL method, since the hair cells present around the dermal papilla are stained, it can be seen that apoptosis occurs in the hair follicle cells when the follicles are degenerate (FIG. 8).

Example 5

Distribution of Precursor Caspase-3

Caspases are all produced as precursors, which are cleaved and activated by molecules present upstream of the cascade. Therefore, localization of precursor caspase-3 in the blanket was investigated using an antibody against precursor caspase-3. Using deparaffinized and ethanol-based hydrophilic human scalp tissue sections, using a mouse antihuman precursor caspase-3 antibody (Immunotech) as the primary antibody and a peroxidase-labeled mouse IgG as the secondary antibody, By the avidin-biotin-peroxidase complex method, the site of the presence of caspase-3 in human hair follicles was immunohistochemically stained.

As shown in FIG. 9, immunostaining of precursor caspase-3 was recognized throughout the human follicle throughout follicular epithelial cells. From this, it was thought that follicle epithelial cells always produce caspase-3, and caspase-3 also acts in apoptosis of follicles.

Example 6

Hair Growth Prolongation Effect by Inhibition of Caspase-3 Activity

Since hair follicle epithelial cells containing hair cells always produce caspase-3, when stimulation of apoptosis is transmitted to hair follicles and activation of caspase-3 occurs in hair cells around the hair papilla, these (hair) Apoptosis is induced in the cells, and as a result, the follicles are considered to be degenerate (hair loss).

From this, it is thought that inhibiting the caspase-3 activity and inhibiting apoptosis of hair cells and hairy root cells may impede or slow down the retraction of hair follicles. That is, the effect of extending the hair growth phase by the inhibition of caspase-3 activity is expected. To verify the specific effect, the hair growth phase prolongation effect is determined in the case of adding a substance that inhibits the caspase-3 activity in the human capillary organ culture method, whether the elongation of the hair is promoted or the shape change of the catenary phase is suppressed. Verified.

Penicillin, streptomycin and Fungizone antibiotics were added to Williams E medium (Gibco) and 10 ng / ml hydrocortisone 10 μg / ml insulin, 10 ng / ml sodium selenite and 10 μg / ml trans The medium to which perrin was added was used for culture of human capillary organs using a medium containing insulin and a medium not added as a basal medium.

Using micro shear blades under stereoscopic microscope, hair follicles of growth phase were isolated from human scalp. The isolated blanket was rinsed in basal medium and then measured for length to allow it to sink in insulin containing medium (24 ml of microplate use: 1 ml per well) of air containing 37 ° C., and 5% CO ₂ . Incubated overnight under weather conditions. After measuring the length again, the blankets with elongation of 0.25 mm or more and the shape of the growth phase were maintained were selected, and divided into groups of 10 to 12 so as to make the kidneys even. For each group, after exchange with a medium containing the test substance, the culture was continued under the above gaseous conditions.

The elongation of the blanket was measured over time by inserting a micrometer into the eyepiece portion of the inverted microscope. Photographs of the entire blanket and the ridges were taken by a camera connected to an inverted microscope.

Carbobenzoxoxy-valyl-alanyl-β-methyl-aspart-1-yl-fluoromethane (z-VAD-fmk) is known to inhibit almost all caspases. Therefore, the hair growth phase extension effect of z-VAD-fmk was verified by the organ culture method. The z-VAD-fmk (final concentration 10, 20, 100 μM) dissolved in DMSO or DMSO of the control group was exchanged with the medium added to the basal medium, and culture was continued while observing elongation and morphological changes of the follicles for 7 days. . As shown in FIG. 10, hair extension was promoted by the addition of z-VAD-fmk compared to the DMSO addition of the control group. Moreover, with the addition of z-VAD-fmk, the proportion of the blanket in which the shape of the hairball portion was maintained increased (Table 2).

Proportion of retained blanket Experiment 1 (Day 6) Experiment 2 (day 4) Control 20% (2/10) 12.5% (1/8) z-VAD 10 μM 37.5% (3/8) z-VAD 20 μM 60% (6/10) z-VAD 100 μM 50.0% (4/8)

Example 7

Screening of Caspase-3 Inhibitors

Substrate Ac-DEVD-MCA (acetyl-Asp-Glu-Val-Asp-methylcoumarinamide) was added to 25 mM HEPES buffer (pH 7.5) containing 10% sucrose, 0.1% CHAPS and 5 mM DTT. 2 μl of the test sample and 2 μl of the caspase-3 enzyme solution were added to 16 μl of the solution, and the reaction mixture of 20 μl in total was incubated at 37 ° C. for 30 minutes, and 200 μl of the reaction stopper was added. Excitation wavelength 355 nm; emission wavelength 460 nm).

Samples to be tested include: kwacharate, comfrey, indigofera tinctoria linn. The extract was used. The results are shown in FIG.

Example 8

Apoptosis Inhibitor Screening

Epidermal keratinocytes of neonatal BALB / c mice were cultured in a high nutrient medium (DMEM medium) containing 10% FCS and 5-fold amino acids and vitamins, resulting in large cells with unclear cell boundaries. In predominance, in part, there was a high density of cells covered with keratin. Large cells with no clear cell boundaries were removed by trypsin treatment, and this operation was repeated over several months to isolate the membranous cell population into Pam212 cells.

The Pam212 cells described above were cultured in DMEM + 10% FCS medium containing 10 ng / ml TNFα and 10 μg / ml cyclohexamide (CHX) to form an apoptosis experiment. On the other hand, Pam212 cells generate apoptosis in a medium containing TNF α and CHX, but do not produce apoptosis when cultured in a medium not containing these additives.

As a sample to be tested, it is known that it has a TGF-2β inhibitory effect, a persimmon extract, a comfrey extract, a maple leaf extract, a quacharrate extract, a mixture of a persimmon extract and a compliment extract, a mixture of a persimmon extract and a maple leaf extract, and a persimmon extract And a mixture of quacharrate extracts. In addition, the test sample was not added to the culture system and the test sample was not added, and the test sample was added to the culture system without adding TNFα and CHX, respectively. Control "," reduction control ", and" quacharate control ". The results are shown in Table 3.

Inhibition of Apoptosis by Pam212 Test sample % Of apoptotic cells Persimmon Extract + Comfrey Extract 100 Persimmon Tea Extract + Maple Leaf Extract 60 Persimmon Extract + Quacharrate Extract 60 Comfrey Extract 117 Maple leaf extract 55 Persimmon Extract 75 Quacharrate Extract 63 Maple leaf control 5 Reduction Control 5 Quacharate control 7 Control 6 TNF α + CHX control 100

As a result, in Example 6, the apoptosis inhibitory effect was recognized in the maple leaf extract, the quacharrate extract, and the persimmon extract, which are known to have a TGF β2 inhibitory effect, in which caspase-3 inhibitory action was recognized.

For example, in FIG. 14, the upper part is a control of Pam212 cells, and the interruption is in the case of culturing Pam212 cells by adding TNF α and CHX, indicating that apoptosis occurs. The bottom is also the case of adding the quacharrate extract and it can be seen that the apoptosis resulting from the disruption is suppressed by the quacharrate extract.

FIG. 15 shows anti-precursor caspase-3 antibody and anti-incubation after incubation for 6 hours with addition of quacharrate extract or VAD-fmk under the conditions in which apoptosis occurs (addition of TNF α and CHX). -Precursor caspase-3 and activated caspase-3 were detected using an activating caspase-3 antibody. Precursor caspase-3 was detected in almost all cells, whereas activated caspase-3 was not detected. This indicates that the quacharrate extract and VAD-fmk completely inhibit the activation of precursor caspase-3.

Example 9

Combination Effects of Fetuin and Z-VAD-fmk, a Caspase-3 Inhibitor with TGF-β2 Inhibitory Activity

Fig. 13 shows a photograph after a week of culturing a blanket in the presence of a fetuin or a caspase-3 inhibitor Z-VAD-fmk having a TGF-β2 inhibitory effect or both. It can be seen that the form of the blanket is very well preserved when both coexist.

As shown below, the proportion of the blanket showing changes in the shape of the catenary in the presence of both clearly decreased.

Example 10

Confirmation of TGF-β2 Promoting the Beginning of the Catenary

(1) Confirmation test in which TGF-β2 promotes the onset of retrogression

Three antibiotics of penicillin, streptomycin and Fungizone were added to Williams E medium (Gibco), and 10 ng / ml hydrocortisone, 10 μg / ml insulin, sodium selenate and 10 μg / ml transferrin The medium to which was added was used for culture of human capillary organs using a medium containing insulin and a medium not added as a basal medium.

Using micro shear blades under stereoscopic microscope, hair follicles of growth phase were isolated from human scalp. The isolated blanket was rinsed in basal medium and then measured for length, allowed to sink in insulin-containing medium (using 24 well microplates: 1 ml per well), followed by gas phase of air containing 37 ° C., 5% CO ₂ . Incubated overnight under conditions. After measuring the length again, the blankets with elongation of 0.25 mm or more and the shape of the growth phase were maintained were selected, and divided into groups of 10 to 12 so as to make the kidneys even.

Each group was exchanged with a medium containing a test substance and then cultured under the above gaseous conditions. The elongation of the blanket was measured over time by inserting a micrometer into the eyepiece portion of the inverted microscope. Photographs of the entire blanket and the ridges were taken by a camera connected to an inverted microscope. On the second day of culture, TGF-β2 was replaced with a medium in which a final concentration of 50 µg / ml was added to the basal medium, and culture was continued while observing elongation and morphological changes of the follicles for 5 days.

On day 6, the follicles to which TGF-β2 had been added promoted the degenerative shape change compared to the control group. In other growth factors and cytokines, the above phenomenon in which the change in the shape of the catenary phase is promoted was not recognized. Therefore, it was thought that TGF-β2 has a catenary action.

In addition, the morphological changes of the hair follicles during the transition from the growth phase to the regression phase in natural hair were also observed. From the comparison of the observation results of both, it is clear that TGF-β2 promotes the transition to the retrograde stage.

(2) Confirmation test of localization of TGF-β2 in human blanket

Human scalp tissues or organ cultured hair follicles were washed with PBS, fixed with 4% paraformaldehyde-phosphate buffer (pH 7.2) for 4 hours, dehydrated with ethanol series, wrapped with paraffin, and then 5 mm thick tissue. Made an intercept.

In order to elucidate the role of TGF-β2 in human hair follicles, localization of TGF-β2 in human hair follicles was investigated. Localization of TGF-β2 in human hair follicles was performed by anti-TGF-β2 antibody immunostaining of human scalp tissue sections.

Using deparaffinized and ethanol hydrophilic sections, anti-TGF-β2 antibody (Santa Cruz) as the primary antibody and peroxidase-labeled rabbit IgG as the secondary antibody, avidin-biotin- By the peroxidase complex method, the site of presence of TGF-β2 in human hair follicles was immunohistochemically stained. As a result, TGF-β2 (immunostainability) was recognized in the outermost layer of the hairy root of the hair follicle in the late growth phase. On the other hand, TGF-β2 (immunostaining staining) was recognized in the hair follicles of the late degenerative phase by the cells of the outermost layer of the outer root sheath and the degenerating epithelial cells of the upper dermal papilla. From this, it was thought that TGF-β2 acts in the late stage of retrogression.

(3) Confirmation test of localization of TGF-β2 in the early stage of degeneration of human blanket

In organ culture of follicles, when the follicle fragments of the growth phase are incubated with an insulin-containing medium, the form of the growth phase is maintained for about two weeks. When the follicles are cultured with a basal medium containing no insulin, the follicles are degenerated. It is changed into a form similar to the blanket of.

Therefore, by culturing the growth phase follicles in a basal medium containing no insulin and examining the localization of TGF-β2 in the follicles showing a slight morphological change, it is possible to estimate whether TGF-β2 is acting in the degenerative phase. Thus, after organ culture for one day, the hairs maintaining the morphology of the growth phase and those showing a slight morphological change similar to those of the degenerative phase were immunostained with anti-TGF-β2.

As a result, TGF-β2 (immunostaining staining) was hardly recognized in the hair and the nipple of the hair follicles, which had maintained its full growth phase.

On the other hand, in the follicles showing a similar morphological change to the degenerative follicles, strong immunostaining of TGF-β2 was recognized at the boundary between the hair and the papilla. From this, it became clear that TGF-β2 also acts in vitro and induces a degenerative phase.

Example 11

Identification of inhibitory effect on hair retrograde transition by TGF-β2 inhibition

From the above results, it has been shown that in the human mother cycle, TGF-β2 acts to induce the degenerative phase. In other words, if the action of TGF-β2 is inhibited in the growth of the hair follicle, it is thought that the transition to the regression phase may be prevented or slowed down. That is, the hair growth phase extension effect by TGF-β2 inhibition can be expected. Specifically, in the case of adding a substance that inhibits the action of TGF-β2 in the culture of human hair follicles, the effect of inhibiting the hair degeneration phase transition is verified by whether hair growth is promoted or the shape change of the degeneration phase is suppressed. did.

(1) Confirmation test of effect of TGF-β neutralizing antibody

The inhibitory effect on hair degenerative transition of TGF-β neutralizing antibodies (known to have neutralizing action of human TGF-β1 and TGF-β2) was verified. The method followed the human blanket organ culture method. On the second day of culture, a medium in which anti-TGF-β antibody (Genzyme Co., Ltd.) or a mouse IgG of the control group was added to the basal medium to a final concentration of 20 µg / ml or 100 µg / ml The culture was continued while observing elongation and morphological changes of the blankets for 4-7 days.

According to this, the addition of TGF-β neutralizing antibody tended to promote hair extension. In addition, as shown in Table 4, the ratio of the hair follicles in which the hairball portion was maintained was increased by the addition of the TGF-β neutralizing antibody.

Proportion of retained blanket Experiment 1 (day 8) Experiment 2 (day 5) Control (mouse IgG) 36.4% (4/11) 55.6% (5/9) 20 μg / ml anti-TGF-β antibody 55.5% (6/11) 100 μg / ml anti-TGF-β antibody 77.8% (7/9)

(2) Test to determine the effect of cyanosaccharide protein fetuin

The cyrosaccharide protein fetuin (molecular weight 48,400) is a substance present in mammalian fetal serum or in adult blood of various diseases (especially during trauma). Since fetuin has an amino acid sequence similar to that of TGF-β and a secondary structure and acts as an antagonist of TCF-β ( Demetriou Met al: Biol Chem 271: 12755-12761, 1996 ), fetuin 's hair degenerative transition The inhibitory effect was verified.

Pettuin (final concentration 1, 10, 50 μM) or control bovine serum albumin (final concentration 50 μm) was exchanged with the medium added to the basal medium and the culture continued for 7 days while observing elongation and morphological changes of the follicles. did. Compared to the control group bovine serum albumin addition, fetuin addition significantly promoted hair growth. From the results of these TGF-β neutralizing antibodies and fetuin, the effect of inhibiting hair degeneration phase by inhibiting TGF-β2 action was demonstrated.

Example 12

Confirmation that the inhibitory effect of TGF-β can be measured by the measurement of PAI-1

Human dermal papilla cells were collected in 96-well plates (about 7,500 cells per well) using DMEM medium containing three kinds of antibiotics, penicillin, streptomycin, and fungizone, in which 10% fetal bovine serum was added. Incubated. First, the TGF-β2 concentration dependency of elevated expression of PAI-1 was investigated. Where appropriate cell density was reached, the cells were exchanged with a medium containing various concentrations of TGF-β2 and incubated for 24 hours. 10 µl of the medium was taken and quantified by a commercially available PAI-1 measurement kit (TintElize PAI-1: biopool). As a result, an increase in PAI-1 was observed in a concentration-dependent manner of TGF-β2.

In addition, when 20 µg / ml TGF-β neutralizing antibody was added at the same time as TGF-β2, the increase of PAI-1 by TGF-β2 was completely suppressed. This result is an example in which the increase of PAI-1 is suppressed when TGF-β and a substance which cancels its action coexist, and this evaluation method is suitable for the screening of the TGF-β action inhibitory substance.

Example 13

Identification test for ingredients in persimmon extract

The present inventors prepared 50% ethanol extract of the persimmon extract, and analyzed the content thereof. As a result, many substances such as filodulcin, hydrangenol, chlorogenic acid, and umbelliferone exist. I confirmed that. The present inventors performed the identification work as described above, but it is already known that these substances are present in the sag extract as shown in FIG.

Subsequently, the present inventors investigated whether or not there is an action of inhibiting the expression of PAI-1 caused by TGF-β2 with regard to filodulcin, an isocoumarin compound. Filodulcin was dissolved in DMSO at a concentration of 0.05-50 μg / ml and used. After 24 hours of incubation with a medium containing 1 ng / ml TGF-β2, the amount of PAI-1 was measured using 10 µl of the medium. In addition, the survival rate of the cells was measured by the Alamar Blue method at the end of the culture.

It was as follows. That is, the cells were washed twice with fresh medium, and then exchanged with a medium containing 1/10 amount of Alama Blue (Alama Bioscience Co., Ltd.) and incubated at 37 ° C (5% CO ₂ ) for 4 hours. . After incubation, the absorbances of 595 nm and 570 nm were measured with a microplate reader to calculate the alamar blue reduction rate of each well, which was divided by the alamar blue reduction rate of the negative control without pilofulcin sample, resulting in cell viability. Calculated.

The former result is shown in FIG. 17, whereby pilodulcin suppresses the increase in expression of PAI-1. That is, the vertical axis | shaft of this figure is an amount of PAI-1, and when it adds pilodulcin, it is 18, it is significantly lower than 100 in the case of no addition, and it can understand that it has the outstanding suppression effect of PAI-1.

18 shows the cytotoxicity of the latter filodulcin, and accordingly, cytotoxicity was hardly recognized in pidululsin. That is, in Fig. 3, the vertical axis represents the alamar blue reduction rate (%). As the value is closer to 100%, the cytotoxicity is lower, and the reduction rate in the case of addition of pilodulcin is 95%. Since the value is close to%, it can be seen that the cytotoxicity is low.

(2) Confirmation test of blanket elongation effect

Verification of the effect of promoting hair growth in the organoculture method of pilodulcin was performed as follows. Filodulcin (wakoponyaku: standard for herbal assays) was dissolved at a concentration of 50 mg / ml DMSO. The method used was in accordance with the human blanket organ culture method. On the second day of culture, the medium was replaced with a medium in which a pilodulcin solution was added at a final concentration of 0.06 µg / ml, and culture was continued while observing the morphology of the kidneys for 7 days.

As a result, it is shown in FIG. 19. According to this, hair extension was significantly promoted in the addition group of pilodulcin 0.06 µg / ml on the 8th day of culture compared to the solvent control group. From this, it is clear that filodulcin selected as an inhibitor of TGF-β has a hair degenerative inhibitory effect and can confirm the effectiveness of the invention.

(3) Confirmation test of hair extension effect of hair

Hair extension when filodulcin was added to isolated human hair was examined. The results are as shown in the photograph of FIG. 20, and it can be clearly confirmed that the hair is elongated compared to the control group when filodulcin is added from this photograph.

It is already known that TGF-β2 is one of the causes of cirrhosis and nephritis, and since pilodulcin has an inhibitory activity (antagonistic action) on TGF-β2 as described above, its performance as an agent for treating cirrhosis or nephritis It is also clear that In addition, isocoumarin compounds other than pilodulcin also have an inhibitory activity (antagonist action) on TGF-β2 like pilodulcin, and are effective as a sheep's wool, a hair degenerative transition inhibitor, a liver cirrhosis drug or a nephritis agent.

By using caspase-3 inhibition and apoptosis inhibition as indicators, screening of hair loss inhibitors can be performed efficiently and with high accuracy.

In addition, novel caspase-3 inhibitors are provided by using caspase-3 inhibition (including apoptosis inhibition, if necessary) as an index.

The present invention shows that tumor growth factor (TGF-β2) promotes the transition from the growth phase to the degenerative phase in the hair growth cycle, and thus obtains the hair growth effect by inhibiting the transition to the regression phase by the antagonist of TGF-β2. The novel screening method developed on the basis of the inventor's findings found that isocoumarins containing pilodulcin have characteristics as active ingredients of wool, hair degenerative transition inhibitors, liver cirrhosis treatment agents, or nephritis medications. It was developed.

The present invention can be effectively expressed as a wool, hair degenerative transition inhibitor, cirrhosis therapeutic agent or kidney therapeutic agent by containing isocoumarins containing pilodulcin in these compositions. In addition, isocoumarins have little cytotoxicity and can exhibit appropriate performance as a wool coat or hair degenerative transition inhibitor. In addition, this compound is also a component in the persimmon tea used for a beverage, and is also an active ingredient which can be used safely in this respect.

Claims (16)

(1) inhibitors against 5α reductase type 2 [5αR-II]; (2) inhibitors to transforming growth factor β2 (TGF β2); And (3) inhibitors to caspase-3 At least two of them are blended with a wool agent. The woolen agent according to claim 1, wherein an inhibitor for transforming growth factor β2 (TGF β2) and an inhibitor for caspase-3 are combined. The woolen agent according to claim 2, wherein the inhibitor against TGF-β2 is a persimmon extract, and the inhibitor against caspase-3 is a maple leaf extract or a quacharrate extract. The method of claim 2, wherein the inhibitor against TGF-β2 is Fetuin and the inhibitor against caspase-3 is Z-VAD-fmk (carbobenzoxy-valyl-alanyl-β-methyl-aspart- 1-yl-fluoromethane). (1) selecting an inhibitor against 5α reductase type 2 (5αR-II); (2) selecting an inhibitor against transforming growth factor β2 (TGF β2); And (3) selecting inhibitors for caspase-3 A method for screening an inhibitor for male hair loss, characterized by combining at least two types of selection. Indigofera tinctoria Linn., Southern Bansaikachi Extract, Clownfish Extract, Tormentirra Extract, Grape Leaf Extract, Bodhi Tree Extract, Quacharate, Caspase-3 inhibitor, characterized in that the extract, the extract of Maple grass, black tea extract, oolong tea extract, Yulmu extract or root extract as an active ingredient. A wool agent comprising the caspase inhibitor of claim 6. The woolen agent according to claim 7, which is an hair loss inhibitor. A wool material characterized by containing isocmarins as an active ingredient. The wool coat according to claim 9, wherein the isocoumarins are phyllodulcin. Hair degenerative transition inhibitor containing isocmarins as an active ingredient. The hair degenerative transition inhibitor according to claim 11, wherein the isocoumarins are pilodulcin. Cirrhosis treatment agent containing isocmarins as an active ingredient. The agent for treating cirrhosis according to claim 13, wherein the isocoumarins are pilodulcin. A nephritis therapeutic agent containing isokumarins as an active ingredient. The agent for treating nephritis according to claim 15, wherein the isocoumarins are pilodulcin.